The present invention relates generally to adjuvants, immunogenic compositions, and methods useful for polynucleotide-based vaccination. The present invention provides compositions and methods useful for enhancing immune response, especially the humoral immune response of vertebrates to polynucleotide-based vaccines. In particular, the present invention provides an adjuvant of cytofectin:co-lipid mixture wherein the cytofectin is (xc2x1)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium bromide (GAP-DMORIE).
In the late 1980s, it was discovered that direct intramuscular (i.m.) injection of lipid-DNA complexes results in measurable protein expression, and that xe2x80x9cnakedxe2x80x9d plasmid DNA (pDNA) is taken up and expressed in muscle to a greater extent than lipid-DNA complexes (Felgner, Scientific American, 276(6), 102-106 (1997)).
One of the first applications of pDNA injection technology was the induction of an immune response. In 1991, it was first reported that mice could be immunized against HIV gp120 by i.m. vaccination with gp120 plasmid DNA (Feigner et al., Nature, 349, 351-352 (1991)), and that mice could be protected from a lethal challenge of influenza virus after DNA immunization with influenza nucleoprotein (NP) antigen. Protection obtained after immunization with the highly conserved NP antigen extended across 2 different viral strains (Ulmer et al., Current Opinions In Immunology, 8, 531-536 (1996)). Numerous publications in the field of polynucleotide-based vaccination followed thereafter (e.g., Boyer et al., J. Med. Primatology, 25(3), 242-250 (1996); Boyer et al., Nature Medicine, 3(5), 526-532 (1997); Davis et al., Vaccine, 15(8), 849-852 (1997); Wang et al., Vaccine, 15(8), 821-825 (1997); Agadjanyan et al., Current Topics In Microbiology And Immunology, 226, 175-192 (1998); Heppell et al., Fish and Shelfish Immunology, 8(4), 271-286 (1998); Lodmell et al., Nature Medicine, 4(8), 949-952 (1998); Vanderzanden et al., Virology, 246(1), 134-144 (1998)).
A major problem frequently encountered in the course of polynucleotide-based vaccination is insufficient or suboptimal humoral response. Often, the antigens or immunogens encoded by the polynucleotide are expressed in vivo, but they are not sufficiently immunogenic to raise the antibody titer in the organism to sufficient levels to provide protection against subsequent challenge and/or to maintain the potential for generating therapeutically active antibody levels over extended time periods. To obtain a stronger humoral and/or cellular response, it is common to administer such vaccines in an immunogenic composition containing an adjuvant, a material which enhances the immune response of the patient to the vaccine. Adjuvants are useful generally for improving the immune response of an organism to a particular immunogen and arc commonly included in vaccine compositions to increase the amount of antibodies produced and/or to reduce the quantity of immunogen and the frequency of administration.
A variety of adjuvants have been reported to effect differing levels of immune response enhancement to polynucleotide-based vaccination. Examples of such adjuvant materials include semi-synthetic bacterial cell wall-derived mono-phosphoryl lipid A (Sasaki, S., et al., Infection and Immunity 65(9), 3250-3258 (1997)), small molecule immunostimulators (Sasaki, S., et al., Clin. Exp. Immunol. 111, 30-35 (1998)), and saponins (Sasaki, S., et al., J. Virology 72(6), 4391-4939 (1998)). The immune response from i.m. pDNA vaccination has also been enhanced through the use of cationic lipids (Ishii, N., et al., Aids Res. Hum. Retroviruses 13(16), 1421-1428 (1997)), Okada, E., et al., J. Immunology 159, 3638-3647 (1997); Yokoyama, M., et al., FEMS Immunol. Med. Microbiol. 14, 221-230 (1996); Gregoriadis, G., et al., FEBS Letters 402, 107-110 (1997); Gramzinski, R. A., et al., Molecular Medicine 4, 109-118 (1998); Klavinskis, L. S., et al., Vaccine 15(8), 818-820 (1997); Klavinskis, L. S., et al., J. Immunology 162, 254-262 (1999); Etchart, N., et al., J. Gen. Virology 78, 1577-1580 (1997); Norman, J., et al., in Methods in Molecular Medicine, Vol. 9; DNA Vaccines: Methods and Protocols, D. B. Lowrie and R. Whalen, eds., Chapter 16, pp. 185-196 (1999)). Cationic lipids were originally studied as cytofectins to enhance delivery of pDNA into cells in vitro, however, further development has led to successful specific applications of protein delivery in vivo (Wheeler, C. J., et al., Proc. Natl. Acad. Sci. USA 93, 11454-11459 (1996); Stephan, D. J., et al., Human Gene Therapy 7, 1803-1812 (1996); DeBruyne, L. A., et al., Gene Therapy 5, 1079-1087 (1998)). Accordingly, such cytofectins may be useful for vaccine applications by enhancing delivery of the pDNA into the cells responsible for giving rise to the humoral arm of the immune response, thereby increasing antibody titer levels.
Commonly used adjuvants show low levels of immune response enhancement for vaccination (typically less than 3-fold) and possess undesirable toxicological and manufacturing profiles. In addition, cationic lipids used previously for vaccination show only low levels of humoral enhancement. There is a need for more adjuvant compositions useful for enhancing the immune response of vertebrates to immunization, especially to pDNA vaccination.
The present invention is directed to adjuvant and immunogenic compositions and to methods for the polynucleotide-based vaccination of a vertebrate, to help protect the vertebrate from a disease, to treat a diseased vertebrate, or both. In certain preferred embodiments, the present invention is directed to a method for immunizing a vertebrate by administering to the vertebrate a composition comprising a polynucleotide that encodes for an immunogen, wherein the polynucleotide is complexed with an adjuvant composition comprising GAP-DMORIE. Preferably, the composition may comprise one or more co-lipids. The immunogen-encoding polynucleotide, upon incorporation into the cells of the vertebrate, produces an immunologically effective amount of an immunogen (e.g., an immunogenic protein). The adjuvant composition of the present invention enhances the immune response of the vertebrate to the immunogen.
One aspect of the present invention is an adjuvant composition comprising a mixture of one or more cytofectins and one or more co-lipids, which adjuvant composition is useful for enhancing the humoral immune response of a vertebrate to an immunogen. Preferably, the adjuvant composition includes the cytofectin GAP-DMORIE and one or more co-lipids. Preferably also, the co-lipid is a neutral lipid such as, for example, a phosphatidylethanolamine. More preferably, the co-lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE), and/or 1,2-dimyristoyl-glycero-3-phosphoethanolamine (DMPE). Most preferably, the co-lipid is DPyPE.
Another aspect of the present invention is an immunogenic composition comprising one or more immunogens and an adjuvant composition compromising the cytofectin GAP-DMORIE and one or more co-lipids. In certain embodiments, the source of the immunogen is an immunogen-encoding polynucleotide, such as in the case of a pDNA vaccine. Preferably, in those embodiments, the pDNA or polynucleotide is complexed with an adjuvant composition comprising GAP-DMORIE and one or more co-lipids.
Another aspect of the present invention is a method for immunizing a vertebrate by administering to the vertebrate an immunogenic composition comprising a complex of one or more immunogen-encoding polynucleotides and GAP-DMORIE in an amount sufficient to generate an immune response to the encoded immunogen. Preferably, the immunogenic composition further includes one or more co-lipids such as, for example, DOPE and/or DPyPE. Most preferably, the co-lipid is DPyPE.
The present invention, in contrast to the prior art, is useful for enhancing the humoral immune response of a vertebrate to a polynucleotide-based vaccine, through the use of GAP-DMORIE. Elevation of antibody levels is particularly advantageous in applications where antibody levels from the immunogen-encoding polynucleotide alone are sub-optimal. In a related advantage, if the desired level of antibodies is produced with a given dose of pDNA, the amount of pDNA necessary to reach the predetermined antibody titer level can be reached using a lower pDNA dose. For pDNA vaccination applications, this advantage is important because acceptable vaccination volumes, coupled with functional limits on the concentration of pDNA, define an upper limit on a given vaccine dose. This advantage is particularly beneficial for vaccines containing multiple plasmids, each of which must be present in sufficient quantity to elicit an immune response to its particular transgene.